Currently, it is estimated up to 150-200 million people may be infected Hepatitis C virus (HCV). Chronic HCV infection can lead to a variety of liver-related diseases, such as liver cirrhosis and hepatocellular carcinoma. As a result, HCV is the leading cause of liver transplantation in the United States. There is no HCV vaccine, and drug therapies are usually insufficient. Despite being such an important virus in terms of worldwide health, the viral life cycle is not understood. Therefore, the objective of this proposal is to study the viral assembly and RNA replication of HCV. Specifically, we propose to research the structure and function of two critical HCV proteins: core protein and nonstructural protein 4B (NS4B). The core protein is a small protein that is the building block of the HCV nucleocapsid. The core protein has three different, but linked, functions: it can bind the HCV RNA genome, self assemble into the viral nucleocapsid, and interact with lipids to create a membrane coat around the nucleocapsid. One specific aim of this proposal is to determine a high resolution crystal structure of the core protein. Crystal structures of the HCV core protein would provide a three dimensional structure to which previous genetic studies can be mapped and further studies could be guided. In addition, co-crystal structures of the core protein with RNA and/or lipid detergents would elucidate the binding mode of core protein and would provide a template for rational drug design targeting core self assembly. A second specific aim of this proposal is the structural and functional studies of HCV NS4B. The NS4B protein is an integral membrane protein that creates the membranous web environment in the host cell, which is the site of HCV RNA replication. One focus of this aim is to use a genetic approach, making NS4B chimeras between different HCV genotypes and subtypes and studying the effects on RNA replication as a means towards understanding NS4B function. A second focus would then be studying pseudorevertants for compensatory second-site mutations, which would reveal the network of interactions between the HCV replicase components. Biochemical and cell biological approaches will then be taken to determine if this interaction is a direct protein-protein interaction or biochemical. The last focus of this aim will be to determine a high resolution crystal structure of NS4B in order to further understand the function of this protein. Overall, the goal of this project is to elucidate the roles of core and NS4B in the HCV life cycle. Understanding the precise role of these proteins in the virus life cycle would not only advance knowledge in basic HCV biology, but would illuminate novel targets for therapeutic drug design.